Name: Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis (Video) | JoVE Journal
Uploaded: 2010-12-17T16:00:00.000+00:00
Duration: 10 min 52 s
Description: Learn to prepare mouse pituitary proteins for studying autoimmune hypophysitis using homogenization and centrifugation. This protocol enables researchers to investigate pituitary gland inflammation mechanisms.

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5 minutes

This work was supported by NIH grant DK080351 to PC.

Experimental Protocol
The first step in this experimental protocol is to collect a large number of mouse pituitary glands. The second step is to homogenize the glands to prepare a protein extract. The third step is to emulsify these proteins with an oily mixture.
Step 1: Collection and storage of mouse pituitary glands
The mouse pituitary gland sits at the base of the skull in a depression of the sphenoid bone called the sella turcica, surrounded laterally by the trigeminal nerves and anteriorly by the optic chiasm. The gland is composed of a larger anterior lobe and smaller posterior and intermediate lobes, and weighs on average 1.9 mg. Pituitary glands are collected from mice of mixed strains, sexes, and ages, destined to be euthanized by the animal care facility.
To isolate the pituitary gland, the mouse is euthanized, the skin above the skull dissected, and the skull bone cut open with scissors. The brain is lifted to expose the pituitary gland, which is then isolated from the surrounding dura mater, scooped out of the sphenoid bone and stored in a plastic tube on dry ice.
In general we use 300 mouse pituitary glands for one protein preparation, a number that can be collected by one person in 9 hours.
Once the collection is completed, the tube containing the pituitary glands is stored at -80 &deg;C until ready for the next step.
Step 2. Extraction of mouse pituitary proteins
<ol>
<li>Add the homogenization buffer to a 15-ml Falcon tube (catalog no 352059) kept in an ice bath, using 250 &mu;L of buffer for every 100 pituitary glands collected. Use a buffer of ionic strength similar to the one that is physiologically present inside the cytoplasm of the cell (0.2 M) and of physiologic pH (7.4). We use phosphate buffered saline (0.161 M, pH 7.4), supplemented with a protease inhibitor cocktail (Sigma-Aldrich, catalog no P8340-1ML).</li>
<li>Add the pituitary glands to the buffer just before homogenization, and then place the generator (5-mm diameter) of the Polytron homogenizer at the bottom of the Falcon tube. Homogenize at maximum rpm for 30 seconds, gently moving the Falcon tube up and down while keeping it in the ice bath. Then, rest the homogenate on ice for 1 min. Repeat the homogenization and cooling steps two more times. This gentle homogenization should break the cells and release cytosolic proteins without disrupting the nuclei.</li>
<li>To remove nuclei, unbroken tissue and insoluble materials (such as connective tissue and denatured protein complexes), centrifuge the homogenate at low speed (1000g) for 10 min at 4 &deg;C.</li>
<li>Transfer the supernatant (now called Post-Nuclear Supernatant, PNS) to a new Falcon tube, taking care to leave the pellet undisturbed, and store the PNS on ice.</li>
<li>Resuspend the pellet in the original volume of homogenization buffer (250 &mu;L of buffer for every 100 pituitary glands collected), homogenize as above, centrifuge as above, and then combine the second PNS with the first one.</li>
<li>Aliquot the two pooled PNS samples into microcentrifuge tubes and store at -80 &deg;C until ready for the next step. This protein preparation can be stored at -80 &deg;C but gradually looses its potency, so we recommend to use it within 6 months from the preparation. Use a small aliquot to determine the protein yield and concentration by the BCA assay (Pierce catalog no. 23225), and the protein quality by gel electrophoresis. In general, we obtain about 60 mg of protein from 300 mouse pituitaries in 1.5 mL of homogenization buffer, at a concentration around 40 mg/ mL.</li>
</ol>
Step 3. Preparation of the emulsion for immunization
<ol>
<li>Thaw rapidly the frozen protein aliquots needed for the experiment and adjust the protein concentration to 20 mg/ mL. This protein extract will be emulsified 1:1 with Complete Freund's Adjuvant (CFA, Sigma F-5881) containing 5 mg/ mL of heat killed Mycobacterium tuberculosis (Becton Dickinson, 231141). Each mouse will receive 1 mg of pituitary extract in 100 &mu;L of emulsion.</li>
<li>The example given here is for immunizing 10 mice, where each mouse will be injected with 100 &mu;L of the emulsion containing 1 mg of pituitary proteins. Allow two extra mice for material loss during the preparation, so use 12 mg of pituitary proteins. Aspire 600 &mu;L of the 20 mg/ mL pituitary protein extract describe above using a 2.5 mL Hamilton gastight syringe. Resuspend the CFA by shaking, and aspire 600 &mu;L of CFA into another 2.5 mL Hamilton syringe. Attach and secure a 22-gauge micro-emulsifying needle to the syringe filled with CFA. Slowly push the plunger of the CFA syringe such that the needle is filled with CFA, then attach and secure the other end of the needle to the syringe filled with pituitary extract.</li>
<li>Slowly advance the plunger of the CFA syringe to push the oil component into the acqueous pituitary extract, such that only a small amount is mixed. Then push back the plunger of the pituitary extract syringe into the CFA syringe. Repeat this action gradually increasing the amount of material mixed. Continue until the entire content of the two syringes are mixed. You will obtain a whitish and evenly distributed solution that represents the water-in-oil emulsion.</li>
<li>Once the emulsion is formed, repeat the back-and-forth motion at least 500 times to ensure the emulsion is thoroughly mixed. After the final mixing cycle, the 1:1 water-in-oil emulsion is ready to use and contains a pituitary protein concentration of 10 mg/mL. The emulsion will be injected the same day subcutaneously into anesthetized SJL mice, as detailed in the companion JoVE article.</li>
</ol>

<ol><li>Caturegli, P., Newschaffer, C., Olivi, A., Pomper, M. G., Burger, P. C., Rose, N. R. Autoimmune Hypophysitis Endocr Rev. 26, 599-614 (2005).</li>
<li>Blansfield, J. A., Beck, K. E., Tran, K., Yang, J. C., Hughes, M. S., Kammula, U. S., Royal, R. E., Topalian, S. L., Haworth, L. R., Levy, C., Rosenberg, S. A., Sherry, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+T-lymphocyte-associated+antigen-4+blockage+can+induce+autoimmune+hypophysitis+in+patients+with+metastatic+melanoma+and+renal+cancer%5BTitle%5D)+AND+%22J+Immunother%22%5BJournal%5D)">Cytotoxic T-lymphocyte-associated antigen-4 blockage can induce autoimmune hypophysitis in patients with metastatic melanoma and renal cancer.</a> J Immunother. 28, 593-598 (2005).</li>
<li>Tzou, S. C., Lupi, I., Landek, M., Gutenberg, A., Tzou, Y. M., Kimura, H., Pinna, G., Rose, N. R., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoimmune+Hypophysitis+of+SJL+mice%3A+Clinical+Insights+from+a+New+Animal+Model%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Autoimmune Hypophysitis of SJL mice: Clinical Insights from a New Animal Model.</a> Endocrinology. 149, 3461-3469 (2008).</li>
<li>Goudie, R. B., Pinkerton, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anterior+hypophysitis+and+Hashimoto%27s+disease+in+a+woman%5BTitle%5D)+AND+%22J+Pathol+Bacteriol%22%5BJournal%5D)">Anterior hypophysitis and Hashimoto's disease in a woman.</a> J Pathol Bacteriol. 83, 584-585 (1962).</li>
<li>Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autoimmune+hypophysitis%3A+an+underestimated+disease+in+search+of+its+autoantigen%28s%29%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">Autoimmune hypophysitis: an underestimated disease in search of its autoantigen(s).</a> J Clin Endocrinol Metab. 92, 2038-2040 (2007).</li>
</ol>

No conflicts of interest declared.

Despite the fact that the first patient with autoimmune hypophysitis was reported in 19624, the pathogenic pituitary autoantigens remain to be identified5. Animal models can be very useful to identify these autoantigens, aiding the discovery of biomarkers that are translatable to patient care. This article has demonstrated a straightforward procedure for preparing pituitary proteins in a form that is adequate for inducing autoimmune hypophysitis in experimental animals.

<table><tbody><tr><td>15-ml Falcon tubes</td><td></td><td></td><td></td></tr><tr><td>homogenization buffer</td><td></td><td></td><td></td></tr><tr><td>phosphate buffered saline</td><td></td><td></td><td></td></tr><tr><td>protease inhibitor cocktail</td><td></td><td></td><td></td></tr><tr><td>Polytron homogenizer</td><td></td><td></td><td></td></tr><tr><td>BCA assay</td><td>Pierce, Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Complete Freund&amp;rsquo;s Adjuvant (CFA)</td><td>Sigma-Aldrich</td><td>F-5881</td><td></td></tr><tr><td>Mycobacterium tuberculosis</td><td>BD Biosciences</td><td>231141</td><td></td></tr><tr><td>2.5 mL Hamilton gastight syringe</td><td></td><td></td><td></td></tr><tr><td>22-gauge micro-emulsifying needle</td><td></td><td></td><td></td></tr></tbody></table>

preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Autoimmune hypophysitis is characterized by an enlargement of the pituitary gland and a deficiency in one or more pituitary hormones. The causative autoantigens remain unknown. Here, a protocol for preparing mouse pituitary gland proteins for use in studying this disease is demonstrated.First pituitary glands are isolated from euthanized mice. The pituitaries are then homogenized to mechanically break the cells and release cytosolic proteins. The homogenous is clarified by low speed centrifugation and assessed for quantity by colormetric, BCA assay and quality by poly acrylamide gel electrophoresis.Finally, the aqueous protein homogenate is mixed with an oily adjuvant. This mixture is then injected into mice to create a mouse model resembling autoimmune hypophysitis. As shown in the companion video, Four techniques have been described to induce hypophysitis in experimental animals.The technique presented in this article that is the injection of pituitary proteins mix influenza adjuvant is the oldest one dating back to the 1960s. Other approaches have been the injection of soluble glycoproteins from the rubella virus in 1992, the stereotactic injection of an adenovirus directly into the pituitary gland in 2002, and the transplantation pituitary gland from one rat strain under the kidney capsule of another rat strain in 2010. The main advantage of the Florence's adjuvant approach is that it is less invasive nowadays, more consistent and above all, more accurate in inducing a disease that closely resembles the human counterpart.Although being reported since the 1960s, the foreign adjuvant technique performed poorly for the induction of hypophysitis. When I started my PhD work, I could obtain only modest hypophysitis instance and severity using the published foreign adjuvant protocol. Things improve in significantly when I have an idea of increasing by tenfold the amount of pitu proteins contain in the EM emotion.Though this methods can provide insights into the pathogenesis of autoimmune hypophysitis, it can also be used to study other autoimmune diseases for which the auto antigens remain to be identified. Generally, individuals new to this methods will struggle mainly with the last step, the preparation of the margin, because it requires manual skills, time and dedication. Visual demonstration is therefore critical to understand how a proper emulsion should be prepared.The implication of this technique extend beyond the field of auto immunity. Knowing how to prepare the in antigen for immunization can also be used for in the production of monoclonal antibodies or for testing new vaccines. Demonstrating this technique will be S Zoo heroic Ura and Mayland al gato from my laboratory.The mouse pituitary is a cylindrical shaped gland that sits at the base of the skull in a depression of the sphenoid bone called the terica. The gland is bordered laterally by the trigeminal nerves and anteriorly by the optic nerve chiasm. It is composed of a larger anterior lobe and smaller, intermediate, and posterior lobes.To isolate the pituitary gland, use scissors to decapitate the euthanized mouse, then make parallel cuts from the back to the front of the skull. Lift the brain and flip it backward to expose the pituitary gland, which appears as a cylinder in the context of an isosceles triangle. Having the pituitary gland as a base, the trigeminal nerves as sides, and the optic chiasm as a vertex.Circle the gland with the tips of closed forceps to break the surrounding meninga. Then using fine forceps, grab the pituitary from the cell of Turca and place it in a plastic tube on dry ice. For one preparation, collect 300 mouse pituitary glands.This collection requires approximately nine person hours. Once the collection is completed, store the pituitary glands at minus 80 degrees Celsius until they're needed. Place a 15 milliliter Falcon tube in an ice bath.Add to the tube 250 microliters of homogenization buffer. For every 100 pituitaries collected just before homogenization, add the mouse pituitary glands to the buffer. Next place the tip of the poly tron homogenizer a five millimeter diameter generator at the bottom of the Falcon tube containing the mouse pituitaries and buffer homogenize at maximum speed for 30 seconds.Gently moving the tube up and down while keeping it on ice. Then rest the homogenate on ice for one minute. Repeat the homogenization and cooling steps two more times.This gentle homogenization should break the cells and release the proteins without breaking the nuclei Centrifuge. The homogenate at low speed, about 1000 G for 10 minutes at four degrees Celsius to sediment, the nuclei, unbroken tissue, and insoluble materials such as connective tissue and denatured protein complexes. Taking care not to disturb the pellet.Transfer the SNA now called post nuclear sate or PNS to a new Falcon tube store. The PNS on ice retract the pellet using the same volume of homogenization buffer as before homogenize and centrifuge as before. After pooling the two PNS samples, eloqua the solution into numerous micro centrifuge tubes.Store the micro centrifuge tubes at minus 80 degrees Celsius until they're needed. This protein preparation is stable at minus 80 degrees Celsius, but gradually loses its potency. Thus, it is recommended to use it within six months from the preparation.Retain one small aliquot of PNS to determine the protein yield and concentration by the biy acid assay. Then determine the protein quality by gel electrophoresis from 300 mouse pituitary glands, which average a total weight of approximately 570 milligrams. Expect a yield of 60 milligrams of pituitary proteins or one milligram of protein for every 10 milligrams of tissue.The protein concentration is typically around 40 milligrams per milliliter. The example provided here is for immunizing 10 mice. Plan to inject each mouse with 100 microliters of emulsion containing one milligram of pituitary protein, allowing two extra mice for material losses during the preparation here, 12 milligrams of protein emulsion are prepared.Begin by rapidly thawing the frozen protein aliquots by warming them up in your hands. Adjust the protein concentration to 20 milligrams per milliliter with PBS. Next, draw 600 microliters of the pituitary protein extract corresponding to 12 milligrams into a 2.5 milliliter Gas Tite Hamilton syringe resuspend, complete FRONS adjuvant or CFA containing five milligrams per milliliter of heat killed mycobacterium tuberculosis by manually shaking it.Then draw 600 microliters of the CFA into another 2.5 milliliter Hamilton syringe. Attach and secure a 22 gauge micro emulsifying needle to the syringe filled with CFA. Slowly push the plunger such that the needle is filled with CFA.Then attach and secure the other end of the needle to the other syringe filled with pituitary protein extract. Avoid trapping air bubbles inside the syringes. Slowly advance the plunger of the CFA syringe to push the oil component into the aqueous pituitary extract such that only a small amount is mixed.Then push back the plunger of the pituitary extract syringe into the CFA syringe. Repeat this action to gradually increasing the amount of material mixed until the entire contents are mixed. This whitish and evenly distributed material represents the water and oil emulsion.Once the emulsion is formed, repeat the full back and forth cycle at least 500 times after the final cycle, the syringes will contain a one-to-one water and oil emulsion where the pituitary protein concentration is now 10 milligrams per milliliter. The emulsion is ready to use to test the quality of the emulsion at a drop of it to a beaker of water. If the emulsion is prepared correctly, the drop will stay compact and tight on the surface.If the emulsion is of poor quality, it will spread and split into smaller drops. The emulsion is injected the same day of its preparation into SJL mice to induce experimental autoimmune hypophysitis. This protocol is detailed in the accompanying jo of vertical.Once the pitu gland had been collected and techniques are mastered, one can perform the protein instruction in one hour, remembering to work on fast and on ice. During this step, During the preparation of the immersion, which requires approximately one hour. Remember to work slowly and patiently in the initial mixing phase After its development.This technique paved the way for researchers in the field of autoimmunity to explore the mechanisms through which autoreactive lymphocytes recognize and infiltrate the target organ. After watching this video, you should have a good understanding on all how to prepare a protein extract from the mouse pituitary gland and how to mix it with Crohn's aju ones to prepare an emulsion that is ready to be used as an immunogen. Don't forget that working with syringes and complete flu edge ones, which contains inactivated and dry microbacterial tuberculosis can be hazardous if you accidentally puncture yourself.So always use G glows and dispose needles properly while performing this procedure.

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Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

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